Human anti-prion antibodies block prion peptide fibril formation and neurotoxicity

Abstract

Prion diseases are a group of rare, fatal neurodegenerative disorders associated with a conformational transformation of the cellular prion protein (PrP(C)) into a self-replicating and proteinase K-resistant conformer, termed scrapie PrP (PrP(Sc)). Aggregates of PrP(Sc) deposited around neurons lead to neuropathological alterations. Currently, there is no effective treatment for these fatal illnesses; thus, the development of an effective therapy is a priority. PrP peptide-based ELISA assay methods were developed for detection and immunoaffinity chromatography capture was developed for purification of naturally occurring PrP peptide autoantibodies present in human CSF, individual donor serum, and commercial preparations of pooled intravenous immunoglobulin (IVIg). The ratio of anti-PrP autoantibodies (PrP-AA) to total IgG was ∼1:1200. The binding epitope of purified PrP-AA was mapped to an N-terminal region comprising the PrP amino acid sequence KTNMK. Purified PrP-AA potently blocked fibril formation by a toxic 21-amino acid fragment of the PrP peptide containing the amino acid alanine to valine substitution corresponding to position 117 of the full-length peptide (A117V). Furthermore, PrP-AA attenuated the neurotoxicity of PrP(A117V) and wild-type peptides in rat cerebellar granule neuron (CGN) cultures. In contrast, IgG preparations depleted of PrP-AA had little effect on PrP fibril formation or PrP neurotoxicity. The specificity of PrP-AA was demonstrated by immunoprecipitating PrP protein in brain tissues of transgenic mice expressing the human PrP(A117V) epitope and Sc237 hamster. Based on these intriguing findings, it is suggested that human PrP-AA may be useful for interfering with the pathogenic effects of pathogenic prion proteins and, thereby has the potential to be an effective means for preventing or attenuating human prion disease progression.

FIGURE 1.

Analysis of PrP_106–126(A117V) binding by purified PrP-AA in an ELISA assay. Purified PrP-AA, non-binding, pass-through IgG (PT) or original IVIg (all at 1 μg) were added to PrP_106–126(A117V) peptide-coated wells. After washing, bound antibodies were detected with horseradish peroxidase-conjugated secondary anti-human IgG antibodies. Purified PrP-AA showed an enhanced signal compared with the original IVIg; whereas, the PT IgG was greatly diminished in binding capacity. E, PrP-AA; PT, pass-through IgG depleted of PrP-AA; IVIg, original IVIg used to purified PrP-AA; **, p < 0.01; ***, p < 0.001.

FIGURE 2.

Characterization of PrP-AA specificity for PrP. A, A117V transgenic mice, but not wild-type (WT) nor PNRP knock-out (KO) mice, were shown to express the PrP protein, which was detectable in brain homogenates using the murine monoclonal antibody 3F4. B, visualization of PrP(A117V) in brain homogenates (500 μg protein) of transgenic mice by immunoprecipitation with purified PrP-AA (E) or PT. Immunoprecipitated complexes were subjected to Western blot analysis with 3F4 antibody. C, purified PrP-AA recognized the PrP protein in Western blots of brain cortex and cerebellar (Cere) homogenates of A117V transgenic mice but not KO mice. Although, multiple bands were observed with overexposure, the strongest signal corresponded to the approximately band of 29 kDa PrP (A117V) observed in PrP(A117V) transgenic mice. D, Western blot analysis of immunoprecipitates from brain homogenates (1 mg transgenic mouse cerebellum and 10 mg Sc237 hamster brain) pretreated with or without proteinase K using PrP-AA or autoantibodies against Aβ. An anti-PrP antibody 6D11 which detects both mouse and hamster PrP, was used for detecting antibody. Numbers adjacent to horizontal lines indicate positions of molecular mass markers (kDa). 10 μl samples were loaded in each lane. Purified PrP-AA recognized both PrP and PK-resistant PrP^Sc (27–30kDa). Autoantibodies against Aβ did not recognize PrP nor PK-resistant PrP^Sc (27–30kDa). The photo was selected from a single representative experiment that was repeated three times with similar results. PT, pass-through IgG depleted of PrP-AA. Aβ-AA, autoantibodies against Aβ

FIGURE 3.

Mapping PrP-AA binding epitopes. Domain specificities of PrP-AA were determined using a peptide microarray. Sequences of either sequentially one amino acids shifted (A) or single amino acids deletions (C) peptides within region PrP_106–126 which were synthesized and spotted on membranes are displayed in A and C. Membranes were then probed with PrP-AA (2 μg/ml) and then HRP conjugated anti-human-IgG antibody (triplicate membranes were probed). The sequence motif KTNMK appeared to be highly important since only peptide 1 is bound by PrP-AA, as shown in panel B. Further validation came from experiments shown in panel D, which show strong binding only when residues 1–5 are present, implying the two lysines (KXXXK) are key elements for binding.

FIGURE 4.

Effects of PrP-AA on PrP peptide's fibril formation. A, dose-response study of PrP_106–126(A117V) fibril formation and PrP-AA effects. B, kinetic study of 50 μm PrP_106–126(A117V) fibril formation and 0.07 μm PrP-AA effects. C, incubation of 50 μm PrP_106–126(A117V) peptides with or without purified PrP-AA in PBS. Purified PrP-AA significantly inhibited PrP_106–126(A117V) fibril formation. D, incubation of preformed fibrils from 50 μm PrP_106–126(A117V) peptides with purified PrP-AA (E, 0.07 μm) or pass-through IgG (PT, 0.07 μm) in PBS for 48 h. Purified PrP-AA significantly disaggregated preformed PrP_106–126(A117V) fibrils as measured by ThT staining. Samples were run in triplicate and plotted as the mean ± S.D. (***, p < 0.001; **, p < 0.01; *, p < 0.05 compared with PrP only, one-way ANOVA). Representative data from triplicate mass spectra of the PrP_106–126(A117V) monomer with (E) or without (F) PrP-AA were inserted to E and F. Electron micrographs of the products from experiments are shown in E and F (scale bar = 500 nm). E, PrP-AA; PT, pass-through IgG depleted of PrP-AA.

FIGURE 5.

Neurotoxicity of PrP peptides on CGN. Dose-dependence of PrP_106–126(A117V) fibril neurotoxicity was examined in CGN. The neurons were exposed to different dosages of PrP_106–126(A117V) (5 μm to 100 μm) (A) or PrP_106–126(A117V) (100 μm) and scrambled control peptide (100 μm) (B) for 3 days. Cell viability was determined by staining neurons with fluorescein diacetate/propidium iodide. Values are expressed as percentages (%) of control (untreated). The data represent the mean ± S.D. (bars) values of triplicate determinations from a single but representative experiment, which has been repeated three times with similar results (**, p < 0.01; ***, p < 0.001 by one-way ANOVA).

FIGURE 6.

Effects of PrP-AA on wild type or mutant PrP_106–126 induced neurotoxicity. Exposure of rat CGN to 50 μm PrP_106–126(A117V or wild type) fibril resulted in a reduction of neuronal survival during a 3 day incubation period. Purified PrP-AA (0.07 μm) significantly attenuated PrP_106–126(A117V) fibril-induced neuronal death. A, PrP_106–126(A117V) peptides (50 μm) were incubated with PrP-AA (0.07 μm) before being exposed to neurons. B, preformed PrP_106–126(A117V) fibrils were incubated with PrP-AA (0.07 μm) before being exposed to neurons. C, preformed wild-type PrP_106–126(117A) fibrils were incubated with PrP-AA (0.07 μm) before exposed to neurons. Cell viability was determined by staining neurons with fluorescein diacetate/propidium iodide. The data represent the mean ± S.D. of triplicate determinations from a representative experiment repeated at least three times with similar results (*, p < 0.05; ***, p < 0.001, compared with PrP_106–126 only, one-way ANOVA). Con, untreated cultures; PrP, PrP_106–126 (A117V or wild type) peptides; E, PrP-AA; PT, pass-through IgG depleted of PrP-AA.

FIGURE 7.

Analysis of PrP-AA in the culture system. The PrP-AA prevented PrP_106–126(A117V) induced neurotoxicity in a neuron-glia co-culture system. Purified PrP-AA significantly blocked PrP_106–126(A117V) fibril-induced neuronal death in the co-cultured system. CGN-glia were treated with 50 μm PrP_106–126(A117V) fibril only and PrP_106–126(A117V) fibril that had been preincubated with 0.07 μm PrP-AA for 24 h. Cell viability was determined by staining neurons with fluorescein diacetate/propidium iodide. Values are expressed as percentages (%) of control (untreated). The data represent the mean ± S.D. (bars) values of triplicate determinations from a single but representative experiment, which has been repeated three times with similar results (**, p < 0.01, by one-way ANOVA). PrP, PrP_106–126(A117V) peptides; E, PrP-AA; PT, pass-through IgG depleted of PrP-AA.